Lumi scint

Manufactured by Bioscan

Sourced in Denmark, United States

Lumi-Scint is a high-performance luminescence detection system designed for sensitive and accurate measurement of luminescent samples. The system utilizes a photomultiplier tube (PMT) detector to capture and quantify light emissions from various types of luminescent assays.

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Lab products found in correlation

Atp standard solution, by Roche (2 mentions) Microrespiration, by Unisense (1 mentions) Rotenone, by Merck Group (1 mentions) Luciferin luciferase chemiluminescent method, by Roche (1 mentions)

6 protocols using lumi scint

Mitochondrial Respiration and ATP Measurement

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O2 consumption was measured at 25 °C in a closed chamber (1.7 ml capacity) using a thermostatically controlled oxygraph apparatus equipped with amperometric electrode (Microrespiration, Unisense A/S, Århus, Denmark) as previously described50 (link).
ATP concentration was measured in a luminometer (Lumi-Scint, Bioscan) by the luciferin/luciferase chemiluminescent method48 . In both cases, 5 mM pyruvate +2,5 mM malate or 20 mM succinate were used as respiring substrates to assess the activity of the pathway formed by Complexes I, III and IV and the pathway composed by Complex II, III and IV, respectively.

Marini C., Ravera S., Buschiazzo A., Bianchi G., Orengo A.M., Bruno S., Bottoni G., Emionite L., Pastorino F., Monteverde E., Garaboldi L., Martella R., Salani B., Maggi D., Ponzoni M., Fais F., Raffaghello L, & Sambuceti G. (2016). Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt. Scientific Reports, 6, 25092.

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Quantitative Measurement of ATP

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ATP concentration was measured in a luminometer (Lumi-Scint, Bioscan) by the luciferin/luciferase chemiluminescent method. The reactions employed are described in supplementary methods.

Marini C., Bianchi G., Buschiazzo A., Ravera S., Martella R., Bottoni G., Petretto A., Emionite L., Monteverde E., Capitanio S., Inglese E., Fabbi M., Bongioanni F., Garaboldi L., Bruzzi P., Orengo A.M., Raffaghello L, & Sambuceti G. (2016). Divergent targets of glycolysis and oxidative phosphorylation result in additive effects of metformin and starvation in colon and breast cancer. Scientific Reports, 6, 19569.

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ATP Synthesis in Rod Outer Segments

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The formation of ATP from adenosine‐3',5'‐diphosfate (ADP) and inorganic phosphate (Pi) was assayed in purified OS (0.005 mg protein/mL) as reported previously. Rod OS were incubated for 10 minutes at 37°C in 50 mmol/L Tris/HCl (pH 7.4), 50 mmol/L KCl, 1 mmol/L ethylene glycol‐bis(beta‐aminoethyl ether)‐N,N,N',N'‐tetraacetic acid, 5 mmol/L MgCl₂, 5 mmol/L KH₂PO₄, 0.6 mmol/L ouabain, and 0.25 mmol/L di(adenosine)‐5‐penta‐phosphate (Ap5A, adenylate kinase inhibitor) in the presence of 20 mmol/L fumarate; 20 mmol/L alpha‐ketoglutarate; 5 mmol/L pyruvate plus 2.5 mmol/L malate; 10 mmol/L citrate; 20 mmol/L beta‐hydroxybutyrate; 0.2 mmol/L NADH; or 20 mmol/L succinate. ATP synthesis was then induced by addition of 0.1 mmol/L ADP and an adequate volume of luciferin/luciferase reagent (Roche Diagnostics Corp.), to continuously follow ATP formation in a luminometer (Lumi‐Scint, Bioscan). ATP standard (Roche Diagnostics Corp.) solutions in the concentration range between 10⁻¹⁰ and 10⁻⁸ mol/L were used for calibration.

Bruschi M., Bartolucci M., Petretto A., Calzia D., Caicci F., Manni L., Traverso C.E., Candiano G, & Panfoli I. (2020). Differential expression of the five redox complexes in the retinal mitochondria or rod outer segment disks is consistent with their different functionality. FASEB BioAdvances, 2(5), 315-324.

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Measuring ATP Synthesis in CT26 Cells

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Fifty micrograms of CT26 cell proteins were incubated in an appropriate buffer plus 5 mM pyruvate and 2.5 mM malate as described in [56 (link)] and ATP synthesis was then induced adding 0.1 mM ADP. ATP concentration was measured in a luminometer (Lumi-Scint, Bioscan) by the luciferin/luciferase chemiluminescent method [57 (link)]. In some experiments, CT26 cells were pre-incubated with 50 μM Rotenone (Sigma) 5 minutes before performing ATP measure. In all experiments the ATP synthesis rate was linear for the first 2 minutes.

Bianchi G., Martella R., Ravera S., Marini C., Capitanio S., Orengo A., Emionite L., Lavarello C., Amaro A., Petretto A., Pfeffer U., Sambuceti G., Pistoia V., Raffaghello L, & Longo V.D. (2015). Fasting induces anti-Warburg effect that increases respiration but reduces ATP-synthesis to promote apoptosis in colon cancer models. Oncotarget, 6(14), 11806-11819.

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Luminescent Measurement of Mitochondrial ATP Synthesis

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An amount of 20 µg of total OS homogenate protein was incubated for 5 min at 37 °C in an assay solution composed by: 50 mM Tris-HCl pH 7.4, 50 mM KCl, 1 mM EGTA, 2 mM MgCl₂, 0.6 mM ouabain, 0.25 mM di(adenosine)-5-penta-phosphate (Ap5A, adenylate kinase inhibitor), and 25µg/mL ampicillin (0.1 mL final volume). As respiratory substrates, 5 mM pyruvate plus 2.5 mM malate were added to the incubation medium. ATP synthesis was induced by the addition of 5 mM KH₂PO₄ and 0.2 mM ADP, at the same pH of the mixture. ATP formation was followed for 2 min in a luminometer (Lumi-Scint, Bioscan, Washington, D.C. USA) by the luciferin/luciferase chemiluminescent method (Roche Diagnostics Corp., Indianapolis, IN, USA). The calibration curve was obtained with ATP standard solutions (Roche Diagnostics Corp., Indianapolis, IN, USA) from 10⁻⁹ and 10⁻⁷ M in the same solution of the experiments [14 (link),31 (link)].

Ravera S., Caicci F., Degan P., Maggi D., Manni L., Puddu A., Nicolò M., Traverso C.E, & Panfoli I. (2020). Inhibitory Action of Antidiabetic Drugs on the Free Radical Production by the Rod Outer Segment Ectopic Aerobic Metabolism. Antioxidants, 9(11), 1133.

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ATP Synthesis in Rod Outer Segments

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The formation of ATP from ADP and inorganic phosphate (Pi) was assayed in purified rod OS (0.04 mg protein/ml) treated as above specified according to Mangiullo et al. [41] and as reported previously (Calzia et al., 2013) (link). Rod OS were incubated for 5 min at 37 °C in 50 mM Tris/HCl (pH 7.4), 5 mM KCl, 1 mM EGTA, 5 mM MgCl 2 , 0.6 mM ouabain, 0.25 mM di(adenosine)-5-penta-phosphate (Ap5A, adenylate kinase inhibitor), and 25µg/ml ampicillin. ATP synthesis was induced by addition of: 5 mM KH 2 PO 4 , 10 mM glucose and 0.1 mM ADP at the same pH of the mixture. After stopping the reaction with 7% perchloric acid final concentration, and neutralizing samples with K 2 CO 3 the ATP concentration was measured in a luminometer (Lumi-Scint, Bioscan) by the luciferin/luciferase chemiluminescent method. Calibration was done with ATP standard solutions (Roche Diagnostics Corp., Indianapolis, IN) between 10 -9 and 10 -7 M.
When necessary, isolated rod OS were preincubated with 30 µM RV for 15 minutes or 5 mM MTF for 2 hours.

Calzia D., Degan P., Caicci F., Bruschi M., Manni L., Ramenghi L.A., Candiano G., Traverso C.E, & Panfoli I. (2018). Modulation of the rod outer segment aerobic metabolism diminishes the production of radicals due to light absorption. Free radical biology & medicine, 117.

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